The emergence of multidrug resistant strains of bacteria is a serious and growing threat to human health. One component of multidrug resistance is the presence of multidrug efflux transporters that expel drugs from the cell thereby keeping their cytosolic levels below toxic concentrations. Nearly all multidrug efflux pump genes are regulated locally by transcription factors that are induced or activated upon binding the very same structurally and chemically dissimilar drugs, which are substrates of the pumps. Thus, these transcription regulators act as intracellular multidrug sensors that increase the number of multidrug efflux pumps when the cell is threatened by an increasing drug dose. Because of their increased solubility, these proteins are outstanding systemsto understand the structural mechanisms of multidrug binding and multidrug gene regulation. In this competing renewal application, x-ray crystallographic studies on two multidrug binding transcription factors, QacR from S. aureus and BmrR from B. subtilis and two multidrug efflux pump gene regulators, MtaN from B. subtilis and MtrR from N. gonorrhoeae, will be done to dissect fully the structural mechanisms by which a single protein can bind multiple structurally and chemically dissimilar drugs as well as regulate the expression of multidrug efflux pump genes. The specific aims are:(1) to determine the x-ray structures of QacR and a series of site directed QacR mutants bound to drugs and DNA: (2) to determine the crystal structures of a number of BmrR-drug-DNA complexes and a series of site directed BmrR mutants bound to drugs and DNA as well as the BmrR-bmr operator complex; (3) to determine the structures of site directed mutants of MtaN in order to elucidate its DNA binding mechanism; (4) to crystallize and determine the structures of MtrR bound to its operator site and off DNA. All specific aims will be supplemented with germane DNA and drug binding studies. These data will be used in future structure-based drug design of novel drugs against pathogenic bacteria, including S. aureus, N. gonorrhoeae and potentially B. anthracis.